The long range goal of the studies described here is to understand the molecular mechanisms and properties of the neuronal cytoskeleton that are essential to the process of neuronal development, aging, and regeneration. Tubulin is a major protein of vertebrate brain and a primary constituent of the neuronal cytoskeleton. Brain microtubules are known to be usually heterogeneous, but the functional significance is not well understood. In this proposal, a specialized form of tubulin and microtubules associated with the axon are characterized by both biochemical and immunochemical methods. This analysis is important for understanding the composition and properties of the axonal cytoskeleton. Previous work has shown that the bulk of axonal tubulin is insoluble and exists in the form of stable domains in axonal microtubules. The distribution of insoluble tubulin in neurons and nonneuronal cells, in subcellular domains, and within microtubules will be analyzed by immunohistochemistry and radioimmunoassays. The role of insoluble tubulin in regulating the organization and stability of the axonal cytoskeleton will be investigated. Since the presence of the stable domains would affect the dynamics of the neuronal cytoskeleton, changes in the axonal cytoskeleton during development, aging, and regeneration are to be examined in detail. Possible mechanisms for regulating properties of the neuronal cytoskeleton in situ, including myelination and neuron-Schwann cell interactions as possible modulators of the neuronal cytoskeleton will be evaluated. These studies will provide a better understanding of the roles that tubulin, microtubules, and the neuronal cytoskeleton play in the development and regeneration of neurons as well as identifying the molecular mechanisms involved in regulating the properties of neuronal microtubules.